The present invention relates to isolated vicilin-like gene promoters, promoter-gene constructs, methods of producing polypeptides in plants, methods of monitoring embryo maturity of conifers, and plants per se.
The expression of seed-storage protein (xe2x80x9cSSPxe2x80x9d) genes in plants is induced during seed development and is restricted to the embryos. SSP gene expression takes place primarily in specialized storage cells located in the embryonic axis, cotyledons, and the endosperm of developing seeds (Goldberg et al., Cell, 56:149-160, 1989). Notably, SSP gene expression does not take place in mature vegetative organs (Thomas, Plant Cell, 5:1401-1410, 1993). In angiosperms and gymnosperms, maximum accumulation of seed-storage proteins and abundant synthesis of oil and starch have been observed during mid to late embryo development.
One example of SSP gene expression is the synthesis of the 2S protein, a seed storage protein encoded by a multi-gene family which synthesis has been shown to be limited to the later stages of seed development. The expression of the 2S protein has been correlated with the accumulation of 2S mRNA (Gander et al., Plant Mol. Biol., 16:437-448, 1991). Regulatory domains within plant promoters found to be necessary for specific expression patterns of SSPs have been determined using deletion and gain of function experiments in transgenic plants (da Silva et al., Plant J., 5:493-505, 1994; de Parter et al., Plant Cell, 5:877-886, 1993; Kawagoe et al., Plant J., 5:885-890,1994; Lessard et al., Plant Mol. Biol., 22:873-885, 1993; Thomas et al., Plant Cell, 2:1171-1180, 1990). Genes that regulate expression of SSPs represent an important model for the study of the mechanisms of developmental-stage and tissue-specific gene expression.
The gene families that encode the main SSPs have been characterized in several plant species (Guerche et al., Plant Cell, 2:469-478, 1990; Higgins, Ann. Rev. Plant Physiol.,35:191-221,1984;Panget al., Plant Mol. Biol., 11:805-820,1988). Seed-storage proteins have been classified into four different groups based on solubility. Albumins, which are water-soluble; globulins, which are salt-soluble; glutelins, which are soluble in acids, alkali ionic detergents, and urea-containing solutions; and prolamins, which are alcohol soluble. The glutelins and prolamins are the major forms of cereal SSPs while the globulins are the most prevalent class of SSPs in legumes and oats (Shotwell et al., The Biochemistry of Plants, Vol. 15: A Comprehensive Treatise, Academic Press, San Diego, Calif., pages 297-345, 1989; Krochko et al., Plant Physiology, 99:46-53, 1992). However, to date there has been no detailed study of the accumulation of seed-storage proteins and the qualitative and quantitative properties of seed-storage proteins in the majority of angiosperms and gymnosperms.
A recent study showed a marked time differential between SSP synthesis and mRNA accumulation during development of zygotic and somatic embryos of alfalfa (Medicago sativa L.). Of three storage proteins studied (7S, 11S, and 2S), mRNA for the 2S protein was found early in somatic embryo development (day 3) but the protein associated with the 2S message was not evident until later (day 10). Thus, both transcriptional and post-transcriptional events appear to be important in determining the protein complement of seed tissues. (Krochko et al., 1992).
Superficially, somatic embryos mimic the developmental stages of zygotic seeds, i.e., the globular, heart, torpedo, and cotyledonary stages (Steward et al., Science, 143:20-27, 1964). However, despite the gross morphological similarities, somatic embryos may exhibit other features which suggest aberrant development, including truncated cotyledonary development, precocious germination, recallusing, multiple or fuse cotyledons, or inability to germinate (Ammirato, Bio/Technology, 1:68-74, 1983; Bapat et al., Plant Cell Rep., 7:538-541, 1988).
Comparisons between somatic and zygotic embryos have shown that somatic embryos can accumulate SSPs (Crouch, Planta, 156:64-74, 1982; Shoemaker et al., Plant Cell Rep., 6:12-15, 1987), and can also be induced to become desiccation tolerant under appropriate culture conditions (Senaratna et al., Plant Sci., 65:253-259, 1989). However, few studies have been conducted on physiological and biochemical changes occurring in somatic embryogenesis in parallel with studies in zygotic embryogenesis. In particular, seed-specific storage proteins, because of their nature and abundance, are expected to be useful markers in the study of gene expression in embryogenic systems.
SSP accumulation is thought to be temporally and spatially regulated, primarily at the level of gene transcription (Muntz, Biochem. Physiol. Pflnazen, 182:93-116, 1987). This conclusion is based primarily on the absence of SSP mRNA in non-seed tissues, and the observed coincidence between the period of maximum seed storage protein synthesis in developing seeds and mRNA accumulation, as determined by Northern blot (Walling et al., Proc. Natl. Acad. Sci. USA, 83:2123-2127, 1986). Factors which affect the accumulation of SSPs in somatic and zygotic embryos vary with the specific SSP, the developmental stage, the tissue (axis or cotyledon), and nutritional conditions (Muntz, Biochem. Physiol. Pflnazen, 182:93-116, 1987; Walling et al., Proc. Natl. Acad. Sci. USA, 83:2123-2127, 1986; Laden et al., Plant Physiol., 84:35-41,1987). The relative magnitude of each of these factors remains to be determined.
Many genes have been introduced into plants using a variety of genetic engineering techniques, sometimes using regulatory elements from sources other than the target plant. Often, the gene coding for the desired polypeptide is placed under the control of a constitutive promoter allowing the expression of the desired polypeptide in the plant throughout the entire life of the plant, irrespective of the plant""s developmental stage. For example, the 35S promoter from Cauliflower Mosaic Virus (CaMV) has been used extensively for constitutive expression of heterologous genes.
For some applications, however, it is not necessary to continuously express the desired polypeptide throughout the life of the plant. In these instances, it is desirable to limit the expression of the desired polypeptide to specific instances, often linked to the stage of plant development. It is often useful to control the time when the desired polypeptide is to be expressed through the use of inducers. In some situations, it is desirable to maintain baseline expression of a desired polypeptide while allowing over-expression of the desired polypeptide at certain times.
While current compositions and methods may be effective to deliver certain genes to plants, there is a need for improved compositions and methods. There is a need for an inducible promoter that will allow the expression of a desired polypeptide. There is also a need for a method of transforming a target plant with a DNA construct comprising an inducible promoter operatively linked to one or more genes coding for desired polypeptides. There is a further need for a method of assessing the developmental maturity of plant embryos.
The present invention is directed to isolated promoters comprising novel nucleic acid sequences. The promoter is a vicilin-like seed storage gene sequence and may be a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The isolated promoter of the present invention includes a fragment thereof having substantially the same activity.
Another embodiment of the present invention is a construct of the promoter operably linked to a desired gene. Also provided by the present invention is a nucleic acid sequence having the functional properties of the promoter of the present invention, the complement of which hybridizes under stringent conditions to the nucleic acid sequence of the promoter.
The present invention is also directed to methods of expressing a desired polypeptide in a plant, the method comprising preparing a construct of a vicilin-like gene promoter sequence operably linked to a desired gene, thereby producing a promoter-gene construct, and transforming a plant with the promoter-gene construct which thereby ultimately expresses a desired polypeptide in the plant. Typically, the polypeptide is a SSP or a structural protein. The desired gene may heterologous or non-heterologous and may be a reporter gene, according to the present invention. The method of expressing a desired polypeptide may include constitutive or induced expression of the desired gene. Suitable inducers include abscisic acid (ABA), heat, and light.
Yet another embodiment of the present invention is a method of monitoring embryo development in conifers, the method comprising preparing a construct of a vicilin-like gene promoter sequence operably linked to a reporter gene, thereby producing a promoter-gene construct; and transforming an embryo with the construct, wherein the expression of the reporter gene is indicative of the degree of activation of the vicilin-like gene promoter thereby providing a qualitative marker of embryo development that may be monitored.
The present invention is also directed to transgenic plants, including transgenic plants comprising a vicilin-like gene promoter operably linked to a desired gene. The plant may be an angiosperm or gymnosperm, preferably a conifer.